Chromatin is the complex of histone proteins, RNA, and DNA that efficiently packages the genome within each human cell. The regulation of chromatin accessibility via post-translational modifications (PTM) of histones is of great current interest as opportunities for pharmacological intervention in the ?writing?, ?reading? and ?erasing? of these PTMs are significant. The biological consequences of most PTMs result from their recruitment of regulatory machinery via protein-protein interactions directly facilitated by the PTM. The binding domains involved in PTM recognition on chromatin are referred to as ?readers?. The overarching objective of this program is to develop an in vivo chemical probe of the CBX reader domains of Polycomb repressive complex 1 (PRC1). There are eight human CBX chromodomains that function as methyl-lysine (Kme) recognition domains (readers) within the two major chromatin repressive complexes that are conserved across higher eukaryotes. CBX proteins 1, 3 and 5 are associated with the heterochromatin protein 1 (HP1) complex. Their Kme binding activity is required for compaction and repression of chromatin that bears the histone H3, lysine 9 trimethyl (H3K9me3) mark. CBX proteins 2, 4, 6, 7, and 8 are associated with the PRC1 which binds the H3K27me3 mark. As appropriate repression of genomic loci is critical throughout organismal development and differentiation, dysregulation of HP1 and Polycomb pathways is implicated in many disease states and an in vivo chemical probe targeting PRC1 would be a first-in-class agent targeting a pathway of high disease relevance and would also represent a unique tool to explore Polycomb biology in complex in vivo systems. The deliverable from this effort will be a high-quality in vivo chemical probe, freely available to the academic community, with confirmed activity and well characterized mechanism versus the CBX readers of PRC1 to catalyze progression of this target toward new therapeutic discoveries in oncology and, potentially, other diseases.